HLA class I-restricted lysis of leukemia cells by a CD8(+) cytotoxic T-lymphocyte clone specific for WT1 peptide

The Wilms tumor (WT1) gene has been reported to be preferentially expressed in acute leukemia cells, regardless of leukemia subtype and chronic myelogenous leukemia cells in blast crisis, but not in normal cells. This finding suggests strongly that WT1 protein is a potential target of immunotherapy for human leukemia. In this study, we established a CD8(+) cytotoxic T-lymphocyte (CTL) clone directed against a WT1-derived peptide and examined its immunologic actions on leukemia cells. A CD8(+) CTL clone, designated TAK-1, which lysed autologous cells loaded with a WT1-derived 9-mer peptide consisting of the HLA-A24 (HLA-A*2402)-binding motifs was established by stimulating CD8(+) T lymphocytes from a healthy individual repeatedly with WT1 peptide-pulsed autologous dendritic cells. TAK-1 was cytotoxic to HLA-A24-positive leukemia cells expressing WT1, but not to HLA-A24-positive lymphoma cells that did not express WT1, HLA-A24-negative leukemia cells, or HLA-A24-positive normal cells. Treating leukemia cells with an antisense oligonucleotide complementary to the WT1 gene resulted in reduced TAK-1-mediated cytotoxicity, suggesting that target antigen of TAK-1 on leukemia cells is the naturally processed WT1 peptide in the context of HLA-A24. TAK-1 did not inhibit colony formation by normal bone marrow cells of HLA-A24-positive individuals. Because WT1 is overexpressed ubiquitously in various types of leukemia cells, but not in normal cells, immunotherapy using WT1 peptide-specific CTL clones should be an efficacious treatment for human leukemia. (Blood. 2000;95:286-293)     

CD8 T-cell responses to Wilms tumor gene product WT1 and proteinase 3 in patients with acute myeloid leukemia

Wilms tumor gene product WT1 and proteinase 3 are overexpressed antigens in acute myeloid leukemia (AML), against which cytotoxic T lymphocytes can be elicited in vitro and in murine models. We performed this study to investigate whether WT1- and proteinase 3-specific CD8 T cells spontaneously occur in AML patients. T cells recognizing HLA-A2.1-binding epitopes from WT1 or proteinase 3 could be detected ex vivo in 5 of 15 HLA-A2-positive AML patients by interferon-gamma (IFN-gamma) ELISPOT assay and flow cytometry for intracellular IFN-gamma and in 3 additional patients by flow cytometry only. T cells producing IFN-gamma in response to proteinase 3 were further characterized in one patient by 4-color flow cytometry, identifying them as CD3(+)CD8(+)CD45RA(+) CCR7(-) T cells, resembling cytotoxic effector T cells. In line with this phenotype, most of the WT1- and proteinase-reactive T cells were granzyme B(+). These results provide for the first time evidence for spontaneous T-cell reactivity against defined antigens in AML patients. These data therefore support the immunogenicity of WT1 and proteinase 3 in acute leukemia patients and the potential usefulness of these antigens for leukemia vaccines.     

Identification of a novel WT1-derived peptide which induces human leucocyte antigen-A24-restricted anti-leukaemia cytotoxic T lymphocytes

We previously reported the establishment of a Wilms' tumour (WT)1-derived peptide (CMTWNQMNL)-specific and human leucocyte antigen (HLA)-A24-restricted anti-leukaemia cytotoxic T lymphocyte (CTL) line, TAK-1. In this study, we have established a novel WT1-derived peptide (RWPSCQKKF)-specific CD8+ CTL line, designated NIM-1. NIM-1 lysed HLA-A24-positive leukaemia cells, but not HLA-A24-negative leukaemia cells or normal cells. The effects of TAK-1 and NIM-1 on cytotoxicity against leukaemia cells were not synergistic, suggesting that recognition of a single epitope on the tumour-specific antigen by CTLs is sufficient to exert maximal cytotoxic activity against tumour cells.     

Redirection of antileukemic reactivity of peripheral T lymphocytes using gene transfer of minor histocompatibility antigen HA-2-specific T-cell receptor complexes expressing a conserved alpha joining region

Donor-derived T lymphocytes directed against minor histocompatibility antigens (mHags) exclusively expressed on cells of the hematopoietic lineages can eliminate hematologic malignancies. Transfer of T-cell receptors (TCRs) directed against these mHags into T lymphocytes may provide a strategy to generate antileukemic T cells. To investigate the feasibility of this strategy the TCR usage of mHag HA-2-specific T-cell clones was characterized. Thirteen different types of HA-2-specific T-cell clones were detected, expressing TCRs with diversity in TCR alpha- and beta-chain usage, however, containing in the TCR alpha chain a single conserved gene segment J alpha 42, indicating that J alpha 42 is involved in HA-2-specific recognition. We transferred various HA-2 TCRs into T lymphocytes from HLA-A2-positive HA-2-negative individuals resulting in T cells with redirected cytolytic activity against HA-2-expressing target cells. Transfer of chimeric TCRs demonstrated that the HA-2 specificity is not only determined by the J alpha 42 region but also by the N-region of the alpha chain and the CDR3 region of the beta chain. Finally, when HA-2 TCRs were transferred into T cells from HLA-A2-negative donors, the HA-2 TCR-modified T cells exerted potent antileukemic reactivity without signs of anti-HLA-A2 alloreactivity. These results indicate that HA-2 TCR transfer may be used as an alternative strategy to generate HA-2-specific T cells to treat hematologic malignancies of HLA-A2-positive, HA-2-expressing patients that received transplants from HLA-A2-matched or -mismatched donors.     

Use of B cell-bound HLA-A2 class I monomers to generate high-avidity, allo-restricted CTLs against the leukemia-associated protein Wilms tumor antigen

Recent studies have detected Wilms tumor antigen (WT1)-specific cytotoxic T lymphocytes (CTLs) in patients with acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML) and demonstrated that most of these CTLs were low avidity. Although HLA-mismatched donors can mount high-avidity CTLs against HLA-A2-presented peptides of WT1, a dominant anti-alloimmune response usually obscures detection of peptide-specific CTLs. Here we explored the feasibility of using recombinant HLA-A2 monomers containing single peptide epitopes as immunogens to generate peptide-specific CTLs from allogeneic donors. We demonstrate that the coating of HLA-A2(-) B lymphocytes with A2/peptide monomers provides a strong stimulus for autologous peptide-specific CTLs. After 3 to 5 rounds of stimulation a population of CD8(+) T cells binding A2/peptide tetramers is easily detectable by fluorescence-activated cell sorting analysis. Furthermore, sorted A2/WT1 tetramer-positive CTLs display strong cytotoxic activity against leukemia cells expressing WT1 endogenously but not against WT1(-) human tumor cells. Thus, HLA/peptide monomers may be useful to isolate peptide-specific donor lymphocytes for treatment of patients with leukemia after HLA-mismatched transplantation.     

Vaccination with an NY-ESO-1 peptide of HLA class I/II specificities induces integrated humoral and T cell responses in ovarian cancer

NY-ESO-1 is a "cancer-testis" antigen expressed in epithelial ovarian cancer (EOC) and is among the most immunogenic tumor antigens defined to date. The NY-ESO-1 peptide epitope, ESO(157-170), is recognized by HLA-DP4-restricted CD4+ T cells and HLA-A2- and A24-restricted CD8+ T cells. To test whether providing cognate helper CD4+ T cells would enhance the antitumor immune response, we conducted a phase I clinical trial of immunization with ESO(157-170) mixed with incomplete Freund's adjuvant (Montanide ISA51) in 18 HLA-DP4+ EOC patients with minimal disease burden. NY-ESO-1-specific Ab responses and/or specific HLA-A2-restricted CD8+ and HLA-DP4-restricted CD4+ T cell responses were induced by a course of at least five vaccinations at three weekly intervals in a high proportion of patients. There were no serious vaccine-related adverse events. Vaccine-induced CD8+ and CD4+ T cell clones were shown to recognize NY-ESO-1-expressing tumor targets. T cell receptor analysis indicated that tumor-recognizing CD4+ T cell clones were structurally distinct from non-tumor-recognizing clones. Long-lived and functional vaccine-elicited CD8+ and CD4+ T cells were detectable in some patients up to 12 months after immunization. These results confirm the paradigm that the provision of cognate CD4+ T cell help is important for cancer vaccine design and provides the rationale for a phase II study design using ESO(157-170) epitope or the full-length NY-ESO-1 protein for immunotherapy in patients with EOC.     

CD4(+) T cell recognition of MHC class II-restricted epitopes from NY-ESO-1 presented by a prevalent HLA DP4 allele: association with NY-ESO-1 antibody production

NY-ESO-1 is a tumor-specific shared antigen with distinctive immunogenicity. Both CD8(+) T cells and class-switched Ab responses have been detected from patients with cancer. In this study, a CD4(+) T cell line was generated from peripheral blood mononuclear cells of a melanoma patient and was shown to recognize NY-ESO-1 peptides presented by HLA-DP4, a dominant MHC class II allele expressed in 43--70% of Caucasians. The ESO p157--170 peptide containing the core region of DP4-restricted T cell epitope was present in a number of tumor cell lines tested and found to be recognized by both CD4(+) T cells as well as HLA-A2-restricted CD8(+) T cells. Thus, the ESO p157--170 epitope represents a potential candidate for cancer vaccines aimed at generating both CD4(+) and CD8(+) T cell responses. More importantly, 16 of 17 melanoma patients who developed Ab against NY-ESO-1 were found to be HLA-DP4-positive. CD4(+) T cells specific for the NY-ESO-1 epitopes were generated from 5 of 6 melanoma patients with NY-ESO-1 Ab. In contrast, no specific DP4-restricted T cells were generated from two patients without detectable NY-ESO-1 Ab. These results suggested that NY-ESO-1-specific DP4-restricted CD4(+) T cells were closely associated with NY-ESO-1 Ab observed in melanoma patients and might play an important role in providing help for activating B cells for NY-ESO-1-specific Ab production.     

Aurora-A kinase: a novel target of cellular immunotherapy for leukemia

Aurora-A kinase (Aur-A) is a member of the serine/threonine kinase family that regulates the cell division process, and has recently been implicated in tumorigenesis. In this study, we identified an antigenic 9-amino-acid epitope (Aur-A(207-215): YLILEYAPL) derived from Aur-A capable of generating leukemia-reactive cytotoxic T lymphocytes (CTLs) in the context of HLA-A*0201. The synthetic peptide of this epitope appeared to be capable of binding to HLA-A*2402 as well as HLA-A*0201 molecules. Leukemia cell lines and freshly isolated leukemia cells, particularly chronic myelogenous leukemia (CML) cells, appeared to express Aur-A abundantly. Aur-A-specific CTLs were able to lyse human leukemia cell lines and freshly isolated leukemia cells, but not normal cells, in an HLA-A*0201-restricted manner. Importantly, Aur-A-specific CTLs were able to lyse CD34+ CML progenitor cells but did not show any cytotoxicity against normal CD34+ hematopoietic stem cells. The tetramer assay revealed that the Aur-A(207-215) epitope-specific CTL precursors are present in peripheral blood of HLA-A*0201-positive and HLA-A*2402-positive patients with leukemia, but not in healthy individuals. Our results indicate that cellular immunotherapy targeting Aur-A is a promising strategy for treatment of leukemia.     

Immunodominant CD4+ responses identified in a patient vaccinated with full-length NY-ESO-1 formulated with ISCOMATRIX adjuvant

There is increasing evidence showing the involvement of CD4(+) T cells in initiating and maintaining antitumor immune responses. NY-ESO-1 is expressed by various tumors but not normal tissues except testis. We conducted a cancer clinical trial by using full-length NY-ESO-1 protein formulated with ISCOMATRIX adjuvant and injected into patients intramuscularly. Autologous dendritic cells pulsed with NY-ESO-1 ISCOMATRIX in combination with overlapping synthetic peptides were used to identify immunodominant T cells from a vaccinated patient. We show here the identification and characterization of two novel CD4(+) T cell epitopes. T cells specific to these epitopes not only recognized autologous dendritic cells loaded with NY-ESO-1 but also NY-ESO-1-expressing tumor cell lines treated with IFN-gamma. One of the two responses identified was greater than the previously identified immunodominant HLA-DP4-restricted response and correlated with NY-ESO-1-specific CD8(+) T cell induction after vaccination. This T cell response was vaccinated in most patients who expressed HLA-DR2. This study has systematically surveyed patients vaccinated with full-length tumor antigen for a vaccinated CD4 helper T cell response.     

Induction of WT1 (Wilms' tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression

The Wilms' tumor gene WT1 is overexpressed in leukemias and various types of solid tumors, and the WT1 protein was demonstrated to be an attractive target antigen for immunotherapy against these malignancies. Here, we report the outcome of a phase I clinical study of WT1 peptide-based immunotherapy for patients with breast or lung cancer, myelodysplastic syndrome, or acute myeloid leukemia. Patients were intradermally injected with an HLA-A*2402-restricted, natural, or modified 9-mer WT1 peptide emulsified with Montanide ISA51 adjuvant at 0.3, 1.0, or 3.0 mg per body at 2-week intervals, with toxicity and clinical and immunological responses as the principal endpoints. Twenty-six patients received one or more WT1 vaccinations, and 18 of the 26 patients completed WT1 vaccination protocol with three or more injections of WT1 peptides. Toxicity consisted only of local erythema at the WT1 vaccine injection sites in patients with breast or lung cancer or acute myeloid leukemia with adequate normal hematopoiesis, whereas severe leukocytopenia occurred in patients with myelodysplastic syndrome with abnormal hematopoiesis derived from WT1-expressing, transformed hematopoietic stem cells. Twelve of the 20 patients for whom the efficacy of WT1 vaccination could be assessed showed clinical responses such as reduction in leukemic blast cells or tumor sizes and/or tumor markers. A clear correlation was observed between an increase in the frequencies of WT1-specific cytotoxic T lymphocytes after WT1 vaccination and clinical responses. It was therefore demonstrated that WT1 vaccination could induce WT1-specific cytotoxic T lymphocytes and result in cancer regression without damage to normal tissues.     

Elimination of human leukemia cells in NOD/SCID mice by WT1-TCR gene-transduced human T cells

Cytotoxic T lymphocytes (CTLs) specific for an HLA-A2-presented peptide epitope of the Wilms tumor antigen-1 (WT1) can selectively kill immature human leukemia progenitor and stem cells in vitro. In this study we have used retroviral gene transfer to introduce a WT1-specific T-cell receptor (TCR) into T lymphocytes obtained from patients with leukemia and from healthy donors. TCR-transduced T cells kill leukemia cells in vitro and display WT1-specific cytokine production. Intravenous injection of TCR-transduced T cells into nonobese diabetic-severe combined immunodeficiency (NOD/SCID) mice harboring human leukemia cells resulted in leukemia elimination, whereas transfer of control T cells transduced with an irrelevant TCR was ineffective. The data suggest that adoptive immunotherapy with WT1-TCR gene-modified patient T cells should be considered for the treatment of leukemia.     

Evidence for Gag p24-specific CD4 T cells with reduced susceptibility to R5 HIV-1 infection in a UK cohort of HIV-exposed-seronegative subjects

AIM: To characterize HIV-1 Gag p24-specific CD4 cell responses in HIV-exposed-seronegative (ES) individuals. METHODOLOGY: Twelve ES individuals, of diverse ethnicity and wild type for the CCR5 Delta-32 mutation, were identified. Controls were HIV-negative blood donors. Gag p24-specific and total Vbeta+ CD4 cells that expressed MIP-1beta, IFN-gamma and IL-2 were enumerated by intracytoplasmic cytokine staining. beta-Chemokine expression was correlated with susceptibility to R5 HIV-1 infection, as measured by polymerase chain reaction for integrated HIV-1 and by p24 enzyme-linked immunosorbent assay. RESULTS: Similar numbers of mitogen-stimulated and Vbeta+ MIP-1beta+, IFN-gamma+ and IL-2+ T cells were found in ES and HIV-negative control subjects. However, all ES subjects tested had an HIV Gag p24-specific MIP-1beta+, IFN-gamma+ and IL-2+ CD4 T-cell response that was rare in controls. p24-Specific cells of all ES but no control subjects could be expanded by in-vitro Ag/IL-2 stimulation, and when re-stimulated with an overlapping peptide series showed evidence of a broad CD4 cell memory response directed against multiple regions of Gag p24. Mitogen-stimulated ES CD4 cells were as susceptible to HIV infection as those from control subjects, but p24-specific IFN-gamma+ CD4 cells of six out of seven ES subjects tested were less susceptible to R5 HIV-1 infection than the counterpart fraction depleted of p24-specific IFN-gamma+ cells. The addition of blocking anti-beta-chemokine antibodies did not promote R5 HIV-1 infection of p24-specific IFN-gamma+ cells. CONCLUSION: Specific CD4 cell immunity, characterized by a broadly directed memory Gag-p24 CD4 cell response and reduced susceptibility of specific CD4 cells to R5 HIV-1 infection, is a likely correlate of non-transmission.     

Hematopoiesis-restricted minor histocompatibility antigens HA-1- or HA-2-specific T cells can induce complete remissions of relapsed leukemia

Donor lymphocyte infusion (DLI) into patients with a relapse of their leukemia or multiple myeloma after allogeneic stem cell transplantation (alloSCT) has been shown to be a successful treatment approach. The hematopoiesis-restricted minor histocompatibility antigens (mHAgs) HA-1 or HA-2 expressed on malignant cells of the recipient may serve as target antigens for alloreactive donor T cells. Recently we treated three mHAg HA-1- and/or HA-2-positive patients with a relapse of their disease after alloSCT with DLI from their mHAg HA-1- and/or HA-2-negative donors. Using HLA-A2HA-1 and HA-2 peptide tetrameric complexes we showed the emergence of HA-1- and HA-2-specific CD8(+) T cells in the blood of the recipients 5-7 weeks after DLI. The appearance of these tetramer-positive cells was followed immediately by a complete remission of the disease and restoration of 100% donor chimerism in each of the patients. Furthermore, cloned tetramer-positive T cells isolated during the clinical response specifically recognized HA-1 and HA-2 expressing malignant progenitor cells of the recipient and inhibited the growth of leukemic precursor cells in vitro. Thus, HA-1- and HA-2-specific cytotoxic T lymphocytes emerging in the blood of patients after DLI demonstrate graft-versus-leukemia or myeloma reactivity resulting in a durable remission. This finding implies that in vitro generated HA-1- and HA-2-specific cytotoxic T lymphocytes could be used as adoptive immunotherapy to treat hematological malignancies relapsing after alloSCT.     

Ti (WT31)-negative, CD3-positive, large granular lymphocyte leukemia with nonspecific cytotoxicity

A case of WT31-, CD3+ large granular lymphocyte leukemia is reported. On surface marker analysis, the proliferating cells were found to be CD3+4-8-16+ and WT31-. By two-color immunofluorescence staining, CD3+4- 8- cells were found to be WT31-, and a small population of WT31+ cells expressed either CD4 or CD8. WT31-, CD3+ cells were also identified in a bulk culture of lymphocytes expanded in vitro. Because WT31 monoclonal antibody (MoAb) reacts with the nonpolymorphic epitope of the disulfide-linked heterodimer of the T cell antigen receptor (Ti), the absence of the WT31-reactive Ti determinant may represent an expression of different CD3-associated polypeptides. The rearrangement of the Ti-beta and Ti-gamma genes but not the immunoglobulin gene was demonstrated, and the single pattern of rearrangement indicated the monoclonal origin of the lymphocytes. When the lymphocytes were assayed for their cytotoxicity against K562, MOLT-4, Daudi, and Raji tumor cell lines, a broad spectrum of cytotoxicity for these tumor cells was observed, and the lymphocytes also exhibited antibody- and lectin- dependent cellular cytotoxicity and lymphokine-activated killer activity. Treatment with anti-CD2 and anti-CD3 MoAbs inhibited their nonspecific cytotoxicity. The anti-CD3-mediated inhibition of nonspecific cytotoxicity suggested that an as yet unidentified Ti, present in association with the CD3 molecule on these lymphocytes, serves as a specific receptor for target tumor cell recognition.     

Specificity for the tumor-associated self-antigen WT1 drives the development of fully functional memory T cells in the absence of vaccination

Recently, vaccines against the Wilms Tumor antigen 1 (WT1) have been tested in cancer patients. However, it is currently not known whether physiologic levels of WT1 expression in stem and progenitor cells of normal tissue result in the deletion or tolerance induction of WT1-specific T cells. Here, we used an human leukocyte antigen-transgenic murine model to study the fate of human leukocyte antigen class-I restricted, WT1-specific T cells in the thymus and in the periphery. Thymocytes expressing a WT1-specific T-cell receptor derived from high avidity human CD8 T cells were positively selected into the single-positive CD8 population. In the periphery, T cells specific for the WT1 antigen differentiated into CD44-high memory phenotype cells, whereas T cells specific for a non-self-viral antigen retained a CD44(low) naive phenotype. Only the WT1-specific T cells, but not the virus-specific T cells, displayed rapid antigen-specific effector function without prior vaccination. Despite long-term persistence of WT1-specific memory T cells, the animals did not develop autoimmunity, and the function of hematopoietic stem and progenitor cells was unimpaired. This is the first demonstration that specificity for a tumor-associated self-antigen may drive differentiation of functionally competent memory T cells.     

Impaired function of human T-lymphotropic virus type 1 (HTLV-1)-specific CD8+ T cells in HTLV-1-associated neurologic disease

Despite abundant activated virus-specific cytotoxic T lymphocytes (CTLs), patients with human T-lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) showed a significantly higher frequency of infected T cells than did healthy virus carriers (HVCs). Here, we demonstrate that at a given proviral load, the frequency of CD8(+) T cells that are negative for specific costimulatory molecules was significantly higher in HAM/TSP than in age-matched HVCs and uninfected healthy controls (HCs), whereas the frequency of intracellular perforin-positive CD8(+) T cells was significantly lower in both HAM/TSP and HVCs than in HCs. An inverse correlation between HTLV-1 proviral load (PVL) and percent perforin-positive CD8(+) T cells were observed only in disease-protective allele HLA-A*02-positive HVCs, but not in HAM/TSP patients, whether HLA-A*02 positive or negative, nor in HLA-A*02-negative HVCs. Significantly lower perforin expression was observed in HTLV-1-specific than in cytomegalovirus-specific CD8(+) T cells. Majority of HTLV-1-specific CD8(+) T cells in HVCs showed a CD28(-)CD27(+) phenotype, whereas HAM/TSP showed a CD28(-)CD27(-) phenotype. HTLV-1-specific CD8(+) T cells from HAM/TSP patients showed significantly lower degranulation than HVCs by CD107a mobilization assay. These findings suggest that an impaired function of HTLV-1-specific CTLs is associated with failing antiviral control and disease HAM/TSP.     

A nonprogressive clinical course in HIV-infected individuals expressing human leukocyte antigen B57/5801 is associated with preserved CD8+ T lymphocyte responsiveness to the HW9 epitope in Nef

The human leukocyte antigen (HLA) B57 allele and the closely related HLA-B5801 allele are overrepresented among human immunodeficiency virus type 1 (HIV-1)-infected individuals with a long-term nonprogressive clinical course of disease (known as "long-term nonprogressors" [LTNPs]). These alleles are, however, also present among individuals with normal disease progression (known as "progressors"). In a comparison of HLA-B57/5801-expressing progressors and LTNPs, we observed a similar prevalence of escape mutations in 4 Nef epitopes and a similar reactivity of CD8+ T cells against 3 of 4 of these epitopes and their autologous escape variants. However, LTNPs tended to have frequent and preserved CD8+ T cell interferon-gamma responses against the wild-type HW9 Nef epitope, whereas progressors did not maintain a specific CD8+ T cell response. This finding is in line with the findings of a more exhausted phenotype of CD8+ T cells in progressors, as is demonstrated by their enhanced level of expression of inhibitory receptor "programmed death 1" (PD-1). The results of the present study suggest that preservation of HW9-specific T cell responses is associated with a more benign clinical course of infection.     

Human dendritic cells presenting adenovirally expressed antigen elicit Mycobacterium tuberculosis--specific CD8+ T cells

Previous studies in murine and human models have suggested an important role for CD8+ T cells in host defense to Mycobacterium tuberculosis (Mtb). Consequently, a successful tuberculosis vaccine may require the elicitation of sustained CD4+ and CD8+ T cell responses. We tested the hypothesis that the potent CD4+ T cell antigen Mtb39 is also a CD8+ T cell antigen. A recombinant adenovirus-expressing Mtb39 (adenoMtb39) was used to infect monocyte-derived dendritic cells. Using interferon-gamma enzyme-linked immunospot, Mtb39-specific CD8+ T lymphocytes were detected in three healthy individuals with latent tuberculosis infection who also had strong anti-Mtb39-specific CD4+ T cell responses. An Mtb39-specific CD8+ T cell line was generated using Mtb39-expressing dendritic cells. Mtb39-specific T cell clones were obtained by limiting dilution cloning. All seven T cell clones obtained were HLA-B44 restricted. Using a panel of synthetic overlapping peptides representative of Mtb39, the peptide epitope was identified for two clones. Furthermore, all T cell clones recognized Mtb-infected dendritic cells and were cytolytic. We conclude that infection of dendritic cells with adenoviral vectors expressing Mtb proteins allows for measurement of antigen-specific CD8+ T cell responses from peripheral blood mononuclear cells. The technique will be useful in defining CD8+ T cell antigens and in measuring immunogenicity of tuberculosis vaccines.